Bioconjugate-nanoparticle probes

ABSTRACT

The invention provides nanoparticle-bioconjugate probes that are useful for detecting target analytes such as nucleic acids. The probes of the invention are stable towards heat and resistant to displacement by thiol containing compounds such as DTT (dithiothreitol).

CROSS REFERENCE

[0001] This application claims the benefit of priority from U.S.Provisional application No. 60/348,239, filed Nov. 9, 2001, which isherein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to stable bioconjugate-nanoparticle probeswhich are useful for detecting nucleic acids and other target analytes.The invention also relates to methods for preparingbioconjugate-nanoparticle probes, to methods of detecting targetanalytes using the probes, and to kits comprising the probes.

BACKGROUND OF THE INVENTION

[0003] The development of methods for detecting and sequencing nucleicacids is critical to the diagnosis of genetic, bacterial, and viraldiseases. See Mansfield, E. S. et al. Molecular and Cellular Probes, 9,145-156 (1995). DNA detection methods that employ gold nanoparticleprobes, modified with oligonucleotides, to indicate the presence of aparticular DNA are described in application number PCT/US00/17507, whichis incorporated by reference herein in its entirety. Typically,oligonucleotides having sequences complementary to the nucleic acid tobe detected are attached to a nanoparticle. The nanoparticle conjugatehybridized to the nucleic acid results in a detectable change resultingfrom the hybridization of the oligonucleotide on the nanoparticle to thenucleic acid target in solution.

[0004] In order to attach the oligonucleotide to the nanoparticle, theoligonucleotide, the nanoparticle or both, are functionalized. Thesemethods are known in the art and include, for instance, thefunctionalization of oligonucleotides with alkanethiols at their3′-termini or 5′-termini. Such functionalized nucleotides readily attachto gold nanoparticles.

[0005] A problem associated with nanoparticles derivatized withalkanethiol-oligonucleotides is that the oligonucleotides are easilydetached from the nanoparticle surface when the system is heated above acertain temperature. Heating destabilizes and inactivates thenanoparticle-oligonucleotide probes. The oligonucleotides can also bedisplaced from the nanoparticle surface in the presence of other thiolcontaining compounds such as DTT.

[0006] There exists a need for oligonucleotide-nanoparticle probes, andbioconjugate-nanoparticle probes in general, that exhibit betteranchoring of the oligonucleotide to the nanoparticle and are thus morestable and robust. Also needed are methods for preparing such complexes.

SUMMARY OF THE INVENTION

[0007] The invention provides a nanoparticle probe comprising abioconjugate of formula (A) coupled to a nanoparticle:

[0008] wherein

[0009] n is 2-100;

[0010] m is 0-100;

[0011] X is a nucleotide, modified oligonucleotide, or a nucleic acidderivative;

[0012] Z is a nucleotide, modified oligonucleotide, or polyanion

[0013] Q is a recognition group. The bioconjugate is coupled to thenanoparticle through the sulfur groups (—SH).

[0014] The invention also provides a nanoparticle probe comprising abioconjugate of formula (B) coupled to a nanoparticle:

[0015] wherein n, m, X, Z and Q are as defined above for bioconjugate(A), and each L is a linker formed by the coupling of two moietiesselected from the group consisting of COOH, NH₂, CHO, Cl, Br, I, NCO,NCS, allyl, and CH3CO₂ ⁻, or L is —C(═NH₂Cl)(CH₂)₃ ⁻. The bioconjugateis coupled to the nanoparticle through the sulfur groups (—SH).

[0016] The invention further provides a nanoparticle probe comprising abioconjugate of formula (C) coupled to a nanoparticle:

[0017] wherein n, m, X, Z and Q are as defined above for bioconjugate(A), and R is an organic moiety such as an alkyl group such linear orbranched C₁-C₈ alkyl, and wherein the bioconjugate is coupled to thenanoparticle through the disulfide groups (—S—S—).

[0018] The invention also provides a nanoparticle probe comprising abioconjugate of formula (D) coupled to a nanoparticle:

[0019] wherein n, m, X, Z, L and Q are as defined above for bioconjugate(B), and wherein W is an aliphatic or aromatic group. The bioconjugateis coupled to the nanoparticle through the sulfur groups (—SH and S—S).

[0020] The invention also provides a nanoparticle probe comprising abioconjugate of formula (E) coupled to a nanoparticle:

[0021] wherein n, m, X, Z and Q are as defined above for bioconjugate(A), and wherein W is an aliphatic or aromatic group. The bioconjugateis coupled to the nanoparticle through the sulfur groups (—SH and S—S).

[0022] The invention also provides a nanoparticle probe comprising abioconjugate of formula (F) coupled to a nanoparticle:

[0023] wherein n, m, X, Z, L and Q are as defined above for bioconjugate(B), and wherein the bioconjugate is coupled to the nanoparticle throughthe sulfur groups.

[0024] The invention also provides a nanoparticle probe comprising abioconjugate of formula (G) coupled to a nanoparticle:

[0025] wherein n, m, X, Z, L, W, R and Q are as defined above. Thebioconjugate is coupled to the nanoparticle through the sulfur groups.

[0026] The invention also provides methods of preparingbioconjugate-nanoparticle probes, methods of detecting target analytesusing the probes and kits comprising the probes of the invention.

[0027] As used herein, a “type of oligonucleotides” refers to aplurality of oligonucleotide molecules having the same sequence. A “typeof” nanoparticles, particles, latex microspheres, etc. havingoligonucleotides attached thereto refers to a plurality of nanoparticleshaving the same type(s) of oligonucleotides attached to them.“Nanoparticles having bioconjugates attached thereto” are also sometimesreferred to as “nanoparticle-bioconjugate probes,” “nanoparticleprobes,” “nano probes,” or just “probes.”

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 depicts detection of an analyte using a substrate.

[0029]FIG. 2 depicts detection of an analyte using a substrate.

[0030]FIG. 3 shows a procedure for introducing amine groups into aoligonucleotide.

[0031]FIG. 4 shows a procedure for introducing thiol groups into aoligonucleotide.

[0032]FIG. 5 shows an alternative procedure for introducing thiol groupsinto a oligonucleotide.

[0033]FIG. 6 depicts a phosphoramidite that can be used for introducingthiol groups into an oligonucleotide.

[0034]FIG. 7 shows a method for preparing an epiandrosterone disulfidederivatized phosphoramidite.

[0035]FIG. 8 shows the incorporation of epiandrosterone disulfide intoan oligonucleotide.

[0036]FIG. 9 shows incorporation of additional thiol groups into anoligonucleotide having a epiandrosterone disulfide moiety.

[0037]FIG. 10 depicts a spot test which indicates binding ofnanoparticle-probes of the invention to a target.

[0038]FIG. 11 depicts spot tests which indicate the relative stabilityof various nanoparticle-probes in dithiothreitol (DTT) solution.

[0039]FIG. 12 depicts spot tests which indicate the relative stabilityof various nanoparticle-probe in DTT solution.

[0040]FIG. 13 depicts spot tests which indicate the relative stabilityof various nanoparticle-probes in DTT solution.

[0041]FIG. 14 depicts spot tests which indicate the relative stabilityof various nanoparticle-probes in DTT solution at elevated temperature.

[0042]FIG. 15 depicts spot tests which indicate the relative stabilityof various nanoparticle-probes in DTT solution, at elevated temperatureand in the presence of magnesium chloride solution.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The bio conjugates of formula (A), (B), (C), (D), (E), (F), and(G) provide a solution to the problem of nanoparticle probe instabilitywhich results when the probe is heated or subjected to thiol containingcompounds. Specifically, the invention permits two or more sulfur groupspresent on a bioconjugate to bind to the nanoparticle surface, whichenhances the stability of the nanoparticle-bioconjugate binding. Theresulting bioconjugate nanoparticle probes are stable towards heat andhave increased resistance to displacement by thiol containing compoundssuch as DTT (dithiothreitol).

[0044] The bioconjugates that are linked to nanoparticles to form thenanop article probes of the invention are of the formulae:

[0045] wherein n, m, X, Z, L, R, W and Q are as defined above.

[0046] As indicated above, Q represents a recognition group. By“recognition group” is meant at least one binding moiety with a bindingaffinity for a target analyte, such as a nucleic acid. Thus, the bindingmoiety may be, for example, one member of a recognition couple whichconsists of two or more substances having a binding affinity of one tothe other. When a bioconjugate is bound to a nanoparticle, therefore, itprovides useful biorecognition properties to the nanoparticle for theanalyte. If the target is a nucleic acid, for example, the recognitiongroup can bind to the nucleic acid by hybridization with the nucleicacid. The nucleic acid bound nanoparticle can then be detected.

[0047] Examples of recognition groups include, without limitation, areceptor, a nucleotide, a nucleoside, a polynucleotide, anoligonucleotide, double stranded DNA, a protein, an antibody, a peptide,a carbohydrate, a sugar, a hapten, a nucleic acid, an amino acid, apeptide nucleic acid, a linked nucleic acid, a nucleoside triphosphate,a lipid, a lipid bound protein, an aptamer, a virus, a cell fragment, ora whole cell. Examples of recognition group-target analyte couplesinclude: an antigen and an antibody; an antigen and an antibodyderivative with a complementary antigen-binding domain; sugar and alectin; a receptor and a ligand; a nucleotide sequence and acomplementary nucleotide sequence; a nucleotide sequence and its bindingprotein or synthetic binding agent; a biotin and avidin or streptavidin;cellulose or chitin and cellulose binding domain. A preferredrecognition group is an oligonucleotide. Also preferred is an antibody.

[0048] The recognition group can also be an oligonucleotide having asequence that is complementary to at least a portion of a secondoligonucleotide having a second recognition group, e.g., anoligonucleotide sequence or protein, bound thereto. The secondrecognition group can then be used for specific binding to a targetanalyte, e.g., an antigen.

[0049] The recognition group can also be a first recognition group,e.g., biotin, that can bind to a second recognition group, e.g.,streptavidin, that is a member of the recognition couple. The secondrecognition group can then be bound directly or indirectly (e.g., via alinker) to a third recognition group, e.g., a receptor, which can bindto a target analyte.

[0050] Z, when present, is a nucleotide spacer, a modifiedoligonucleotide, a polyanion, or other type of spacer which may beutilized in oligonucleotide synthesis such as a polyethylene glycol. Ithas been found that hybridization efficiency ofnanoparticle-bioconjugate probes with nucleic acids can be increased bythe use of a spacer portion between the recognition group on thebioconjugate and the nanoparticle. By using a spacer portion, therecognition group is spaced away from the surface of the nanoparticlesand is more accessible for hybridization with its target. The length andsequence of the spacer portion providing good spacing of the recognitionportion away from the nanoparticles can be determined empirically. Ithas been found that a spacer portion comprising at least about 10nucleotides, preferably 10-50 nucleotides, gives good results. Thespacer portion may have any sequence which does not interfere with theability of the recognition group to become bound to a target analyte ora capture moiety on a surface in sandwich hybridization assays. Forinstance, the spacer portions should not have sequence complementary toeach other, to that of the recognition group, or to that of the targetanalyte. Preferably, the bases of the nucleotides of the spacer portionare all adenines, all thymines, all cytidines, or all guanines, unlessthis would cause one of the problems just mentioned. More preferably,the bases are all adenines or all thymines. Most preferably the basesare all thymines. Spacer Z and recognition group Q can be attachedtogether by a variety of techniques. For instance, they can be attacheddirectly by a covalent linkage or indirectly by non-covalent linkage.

[0051] As a linker, L can be any desired chemical group. For instance, Lcan be a polymer (e.g., polyethylene glycol, polymethylene, protein,peptide, oligonucleotide, or nucleic acid), —COO—, —CH₂(CH₂)_(v)COO—,—OCO—, R¹N(CH₂)_(v)—NR¹—, —OC(CH₂)_(v)—, —(CH₂)_(v)—, —O—(CH₂)_(v)—O—,—R¹N—(CH₂)_(v)—,

[0052] v is 0-30 and R¹ is H or is G(CH₂)_(v), wherein G is —CH₃,—CHCH₃, —COOH, —CO₂(CH₂)_(v)CH₃, —OH, or —CH₂OH.

[0053] L is also a linker formed by the coupling of two moietiesattached to molecules, the moieties selected from the group consistingof COOH, NH₂, CHO, Cl, Br, I, NCO, NCS, allyl, and CH₃CO₂ ⁻, or L is—C(═NH₂Cl)(CH₂)₃ ⁻.

[0054] W is an aliphatic or aromatic group on which sulfur moieties canbe readily bound. For instance, W can be steroid. Preferably W is anepiandrosterone derivative, as described in example 9.

[0055] X represents a nucleotide that has been functionalized with athiol group (as shown). Preferably, the X groups are all adenines, allthymines, all cytidines, or all guanines, more preferably, all adeninesor all thymines, most preferably all thymines. In (X)_(n), n is 2-100.Preferably, n is 2-50, more preferable 2-20, more preferably 2-10.Functionalization of the X linkage with a thiol group can be carried outby a variety of techniques.

[0056] In one embodiment of the invention, either a complex (I) orcomplex (II) containing at least two reactive groups R₂ is synthesizedby incorporating nucleotide building blocks containing an R₂ groupduring synthesis of the oligonucleotide. R₂ can be COOH, NH₂, CHO, F,Cl, Br, I, NCO, NCS, allyl, or CH₃CO₂ ⁻. If R₂ in complex (I) or (II) isan NH₂, then a nucleotide derivative containing an NH₂ group is preparedfor incorporation into the oligonucleotide. Suitable amine modifiednucleotide reagents for use in this aspect of the invention include, butare not limited to, C₆-dT phosphoramidite(5′-Dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2′-deoxyUridine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite),amino modifier C₆-dC(5′-Dimethoxytrityl-N-dimethylformamidine-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2′-deoxy-Cytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite),and amino modifier C₂-dT (5′-dimethoxytrityl-5-[N-(trifluoroacetylaminoethyl)-3-acrylimido]-2′-deoxy-Uridine,3′-[(2-cyanoethyl)-(N,N-di-isopropyl)]-phosphoramidite).These and other amino modifiers are commercially available, for examplefrom Glen Research, Sterling, Va. A preferred amino modifier reagent isC₆-dT.

[0057] Complex (I) or (II) is reacted with a thiolating reagent that isfunctionalized with a group capable of reacting with the R₂ group on thecomplex and resulting in formation of a sulfur functionalizedbioconjugate. Suitable thiolating reagents generally include thiolcompounds possessing one or more functional groups capable of reactingwith the R₂ group of the complex. Such reagents include, but are notlimited to, cystamine, and compounds of the formula SH(CH₂)_(n)Y,wherein n is 1-20 and Y is COOH, NH₂, CHO, F, Cl, Br, I, NCO, NCS,allyl, or CH₃CO₂ ⁻. Another suitable thiolating reagent is2-iminothiolane hydrochloride (Traut's Reagent), which is preferred whenR₂ in complex (I) or (II) is an amine. In a preferred aspect of thisembodiment, fluorinated nucleotides are incorporated into anoligonucleotide sequence and treated with cystamine to provide disulfideunits on the sequence (References (1) L. V. Nechev, I. Kozekov, C. M.Harris, and T. M. Harris, Chem Res Toxicol, 2001, 14, 1506-1512. (2) A.R. Diaz, R. Eritja, and R. G. Garcia, Nucleos Nucleot, 1997, 16,2035-2051. (3) D. A. Erlanson, J. N. M. Glover, and G. L. Verdine, J.Amer. Chem. Soc., 1997, 119, 6927-6928). This preferred aspect isdescribed in detail in Examples 7-9, below.

[0058] An alternative method of thiolating complex (I) or (II) is to usea thiolating reagent that is a combination of reagents. For instance, ifR₂ in complex (I) or (II) is NH₂, then the complex can be treated withan amine reactive bifunctional crosslinker, such as CHO(CH₂)_(n)CHO, andan alkyl or aryl thiol amine, such as SH(CH₂)_(n)NH₂ or SH(C₆H₄)NH₂. Inboth the amine reactive bifunctional crosslinker and the alkyl thiolamine, n is independently 1-30. Preferably, the amine reactivebifunctional crosslinker is glutaraldehyde (i.e., n is 3). Alsopreferably, the alkyl thiol amine is mercaptoethylamine (i.e., n is 2).Other preferred crosslinkers include 1,4 phenylene diisothiocyanate, 1,6dihexanoic acid, or 1,6 hexane diisocyanate.

[0059] In an alternative and more direct approach for preparing a thiolfunctionalized bioconjugate, a phosphoramidite containing an alkylthiolor other thiol based group is synthesized and used to prepare abioconjugate of formula (A). An example of a phosphoramidite containingan alkylthiol group is described in Example 4, below, and is preparedaccording to the method of Glick et al., Tetrahedron Letters, 1993, 34,5549-5552 which is incorporated herein by reference.

[0060] As indicated above, the invention provides bioconjugatenanoparticle probes that are useful for detecting target analytes. Toform the probe, bioconjugates (A), (B), (C), (D), (E), (F), or (G) areconnected to the surface of a nanoparticle through the sulfur linkageson the bioconjugate. Preferably, the connection is through at least twothiol groups per bioconjugate molecule. Various methods can be used toconnect the bioconjugate to the nanoparticle. In fact, any suitablemethod for attaching a bioconjugate to a nanoparticle may be used. Apreferred method for attaching an bioconjugate to a nanoparticle isbased on an aging process described in U.S. application Ser. No.09/344,667, filed Jun. 25, 1999; Ser. No. 09/603,830, filed Jun. 26,2000; Ser. No. 09/760,500, filed Jan. 12, 2001; Ser. No. 09/820,279,filed Mar. 28, 2001; Ser. No. 09/927,777, filed Aug. 10, 2001; and inInternational application nos. PCT/US97/12783, filed Jul. 21, 1997;PCT/US00/17507, filed Jun. 26, 2000; PCT/US01/01190, filed Jan. 12,2001; PCT/US01/10071, filed Mar. 28, 2001, the disclosures of which areincorporated by reference in their entirety.

[0061] The aging process provides nanoparticle-bioconjugate probes withenhanced stability and selectivity. The method comprises providingbioconjugates having covalently bound thereto thiol functional groups,prepared as described above. The functionalized bioconjugates arecontacted with the nanoparticles in water for a time sufficient to allowat least some of the bioconjugates to bind to the nanoparticles by meansof the functional groups. Such times can be determined empirically. Forinstance, it has been found that a time of about 12-24 hours gives goodresults. Other suitable conditions for binding of the bioconjugates canalso be determined empirically. For instance, a concentration of about10-20 nM nanoparticles and incubation at room temperature gives goodresults.

[0062] Next, at least one salt is added to the water to form a saltsolution. The salt can be any suitable water-soluble salt. For instance,the salt may be sodium chloride, lithium chloride, potassium chloride,cesium chloride, ammonium chloride, sodium nitrate, lithium nitrate,cesium nitrate, sodium acetate, lithium acetate, cesium acetate,ammonium acetate, a combination of two or more of these salts, or one ofthese salts in phosphate buffer. Preferably, the salt is added as aconcentrated solution, but it could be added as a solid. The salt can beadded to the water all at one time or the salt is added gradually overtime. By “gradually over time” is meant that the salt is added in atleast two portions at intervals spaced apart by a period of time.Suitable time intervals can be determined empirically.

[0063] The ionic strength of the salt solution must be sufficient toovercome at least partially the electrostatic repulsion of thebioconjugates from each other and, either the electrostatic attractionof the negatively-charged bioconjugates for positively-chargednanoparticles, or the electrostatic repulsion of the negatively-chargedbioconjugates from negatively-charged nanoparticles. Gradually reducingthe electrostatic attraction and repulsion by adding the salt graduallyover time has been found to give the highest surface density ofbioconjugates on the nanoparticles. Suitable ionic strengths can bedetermined empirically for each salt or combination of salts. A finalconcentration of sodium chloride of from about 0.1 M to about 3.0 M inphosphate buffer, preferably with the concentration of sodium chloridebeing increased gradually over time, has been found to give goodresults.

[0064] After adding the salt, the bioconjugates and nanoparticles areincubated in the salt solution for an additional period of timesufficient to allow sufficient additional bioconjugates to bind to thenanoparticles to produce the stable nanoparticle-bioconjugates probes.An increased surface density of the bioconjugates on the nanoparticleshas been found to stabilize the probes. The time of this incubation canbe determined empirically. A total incubation time of about 24-48,preferably 40 hours, has been found to give good results (this is thetotal time of incubation; as noted above; the salt concentration can beincreased gradually over this total time). This second period ofincubation in the salt solution is referred to herein as the “aging”step. Other suitable conditions for this “aging” step can also bedetermined empirically. For instance, incubation at room temperature andpH 7.0 gives good results. The solution is then centrifuged and thenanoparticle probes processed as desired. For instance, the solution canbe centrifuged at 14,000 rpm in an Eppendorf Centrifuge 5414 for about15 minutes to give a very pale pink supernatant containing most of theoligonucleotide (as indicated by the absorbance at 260 nm) along with7-10% of the colloidal gold (as indicated by the absorbance at 520 nm),and a compact, dark, gelatinous residue at the bottom of the tube. Thesupernatant is removed, and the residue is resuspended in the desiredbuffer.

[0065] The probes produced by use of the “aging” step have been found tobe considerably more stable than those produced without the “aging”step. As noted above, this increased stability is due to the increaseddensity of the bioconjugates on the surfaces of the nanoparticles whichis achieved by the “aging” step. The surface density achieved by the“aging” step will depend on the size and type of nanoparticles and onthe length, sequence and concentration of the oligonucleotides. Asurface density adequate to make the nanoparticles stable and theconditions necessary to obtain it for a desired combination ofnanoparticles and oligonucleotides can be determined empirically.

[0066] Oligonucleotides or other recognition elements containingmultiple thiol moieties as described above may bind to a variety ofnanoparticles that have an affinity for thiol groups. Nanoparticlesuseful in the practice of the invention include metal (e.g., gold,silver, platinum, cobalt), semiconductor (e.g., Si, CdSe, CdS, and CdSor CdSe coated with ZnS), core shell particles (e.g., gold coated silverparticles), alloy particles (e.g. silver and gold alloy), magnetic(e.g., cobalt), and non metallic (e.g. silicon) colloidal materials.Core shell particles are described in PCT applications PCT/US01/50825,and PCT/US02/16382, as well as copending U.S. application Ser. Nos.10/153,483 and 10/034,451, each of which is incorporated herein byreference. Other nanoparticles composed of materials that have anaffinity for thiol groups may also be used. In addition, nanowires ornanorods having a composition with an affinity for thiol groups also maybe used. The size of the nanoparticles is preferably from about 5 nm toabout 150 nm (mean diameter), more preferably from about 5 to about 50nm, most preferably from about 10 to about 30 nm.

[0067] Methods of making metal, semiconductor and magnetic nanoparticlesare well-known in the art. See, e.g., Schmid, G. (ed.) Clusters andColloids (V C H, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold:Principles, Methods, and Applications (Academic Press, San Diego, 1991);Massart, R., IEEE Transactions On Magnetics, 17, 1247 (1981); Ahmadi, T.S. et al., Science, 272, 1924 (1996); Henglein, A. et al., J. Phys.Chem., 99, 14129 (1995); Curtis, A. C., et al., Angew. Chem. Int. Ed.Engl., 27, 1530 (1988). Methods of making ZnS, ZnO, TiO₂, AgI, AgBr,HgI₂, PbS, PbSe, ZnTe, CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, andGaAs nanoparticles are also known in the art. See, e.g., Weller, Angew.Chem. Int. Ed. Engl., 32, 41 (1993); Henglein, Top. Curr. Chem., 143,113 (1988); Henglein, Chem. Rev., 89, 1861 (1989); Brus, Appl. Phys. A.,53, 465 (1991); Bahncmann, in Photochemical Conversion and Storage ofSolar Energy (eds. Pelizetti and Schiavello 1991), page 251; Wang andHerron, J. Phys. Chem., 95, 525 (1991); Olshavsky et al., J. Am. Chem.Soc., 112, 9438 (1990); Ushida et al., J. Phys. Chem., 95, 5382 (1992).

[0068] Suitable nanoparticles are also commercially available from,e.g., Ted Pella, Inc. (gold), Amersham Corporation (gold) andNanoprobes, Inc. (gold). Presently preferred nanoparticles are goldnanoparticles.

[0069] The bioconjugate-nanoparticle probes of the invention can be usedto detect target analytes, such as nucleic acids. Examples of nucleicacids that can be detected with nanoparticle probes of the inventioninclude genes (e.g., a gene associated with a particular disease), viralRNA and DNA, bacterial DNA, fungal DNA, cDNA, mRNA, RNA and DNAfragments, oligonucleotides, synthetic oligonucleotides, modifiedoligonucleotides, single-stranded and double-stranded nucleic acids,natural and synthetic nucleic acids, etc. Thus nanoparticle probesprepared according to the invention can be used, for example, for thediagnosis and/or monitoring of viral diseases (e.g., humanimmunodeficiency virus, hepatitis viruses, herpes viruses,cytomegalovirus, and Epstein-Barr virus), bacterial diseases (e.g.,tuberculosis, Lyme disease, H. pylori, Escherichia coli infections,Legionella infections, Mycoplasma infections, Salmonella infections),sexually transmitted diseases (e.g., gonorrhea), inherited disorders(e.g., cystic fibrosis, Duchene muscular dystrophy, phenylketonuria,sickle cell anemia), and cancers (e.g., genes associated with thedevelopment of cancer); in forensics; in DNA sequencing; for paternitytesting; for cell line authentication; for monitoring gene therapy; andfor many other purposes.

[0070] To perform an assay according to the invention, a samplesuspected of containing a target analyte is contacted with bioconjugatenanoparticle probes having attached thereto recognition groups capableof binding to at least a portion of the target analyte. The target to bedetected may be isolated by known methods, or may be detected directlyin cells, tissue samples, biological fluids (e.g., saliva, urine, blood,serum), solutions containing PCR components, solutions containing largeexcesses of oligonucleotides or high molecular weight DNA, and othersamples, as also known in the art. See, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual (2nd ed. 1989) and B. D. Hames and S. J.Higgins, Eds., Gene Probes 1 (IRL Press, New York, 1995). Methods ofpreparing nucleic acids for detection with hybridizing probes are wellknown in the art. See, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (2nd ed. 1989) and B. D. Hames and S. J. Higgins,Eds., Gene Probes 1 (IRL Press, New York, 1995).

[0071] If a nucleic acid is present in small amounts, it may beamplified by methods known in the art. See, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and B. D. Hamesand S. J. Higgins, Eds., Gene Probes 1 (IRL Press, New York, 1995).Preferred is polymerase chain reaction (PCR) amplification.

[0072] One method according to the invention for detecting nucleic acidcomprises contacting a nucleic acid with one or more types ofnanoparticle probes of the invention. The nucleic acid to be detectedhas at least two portions. The lengths of these portions and thedistance(s), if any, between them are chosen so that when thebioconjugates on the nanoparticles hybridize to the nucleic acid, adetectable change occurs. These lengths and distances can be determinedempirically and will depend on the type of particle used and its sizeand the type of electrolyte which will be present in solutions used inthe assay (as is known in the art, certain electrolytes affect theconformation of nucleic acids).

[0073] Also, when a nucleic acid is to be detected in the presence ofother nucleic acids, the portions of the nucleic acid to which thebioconjugates on the nanoparticles are to bind must be chosen so thatthey contain sufficient unique sequence so that detection of the nucleicacid will be specific. Guidelines for doing so are well known in theart.

[0074] Although nucleic acids may contain repeating sequences closeenough to each other so that only one type of bioconjugate-nanoparticleconjugate need be used, this will be a rare occurrence. In general, thechosen portions of the nucleic acid will have different sequences andwill be contacted with nanoparticles carrying two or more differentbioconjugates, preferably attached to different nanoparticles.Additional portions of the DNA could be targeted with correspondingnanoparticles. Targeting several portions of a nucleic acid increasesthe magnitude of the detectable change.

[0075] The contacting of the nanoparticle-bioconjugate probes with thenucleic acid takes place under conditions effective for hybridization ofthe bioconjugates on the nanoparticles with the target sequence(s) ofthe nucleic acid. These hybridization conditions are well known in theart and can readily be optimized for the particular system employed.See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nded. 1989). Preferably stringent hybridization conditions are employed.

[0076] Faster hybridization can be obtained by freezing and thawing asolution containing the nucleic acid to be detected and thenanoparticle-bioconjugate probes. The solution may be frozen in anyconvenient manner, such as placing it in a dry ice-alcohol bath for asufficient time for the solution to freeze (generally about 1 minute for100 microliters of solution). The solution must be thawed at atemperature below the thermal denaturation temperature, which canconveniently be room temperature for most combinations ofnanoparticle-bioconjugate probes and nucleic acids. The hybridization iscomplete, and the detectable change may be observed, after thawing thesolution.

[0077] The rate of hybridization can also be increased by warming thesolution containing the nucleic acid to be detected and thenanoparticle-bioconjugate probes to a temperature below the dissociationtemperature (Tm) for the complex formed between the bioconjugates on thenanoparticles and the target nucleic acid. Alternatively, rapidhybridization can be achieved by heating above the dissociationtemperature (Tm) and allowing the solution to cool.

[0078] The rate of hybridization can also be increased by increasing thesalt concentration (e.g., from 0.1 M to 1 M NaCl).

[0079] The detectable change that occurs upon hybridization of thebioconjugates on the nanoparticles to the nucleic acid may be an opticalchange (e.g. color change), the formation of aggregates of thenanoparticles, or the precipitation of the aggregated nanoparticles. Theoptical changes can be observed with the naked eye or spectroscopically.The formation of aggregates of the nanoparticles can be observed byelectron microscopy or by nephelometry. The precipitation of theaggregated nanoparticles can be observed with the naked eye ormicroscopically. Preferred are color changes observable with the nakedeye.

[0080] The observation of a color change with the naked eye can be mademore readily against a background of a contrasting color. For instance,when gold nanoparticles are used, the observation of a color change isfacilitated by spotting a sample of the hybridization solution on asolid white surface (such as silica or alumina TLC plates, filter paper,cellulose nitrate membranes, and nylon membranes, preferably a nylonmembrane) and allowing the spot to dry. Initially, the spot retains thecolor of the hybridization solution (which ranges from pink/red, in theabsence of hybridization, to purplish-red/purple, if there has beenhybridization). On drying at room temperature or 80° C. (temperature isnot critical), a blue spot develops if the nanoparticle-bioconjugateprobes had been linked by hybridization with the target nucleic acidprior to spotting. In the absence of hybridization (e.g., because notarget nucleic acid is present), the spot is pink. The blue and the pinkspots are stable and do not change on subsequent cooling or heating orover time. They provide a convenient permanent record of the test. Noother steps (such as a separation of hybridized and unhybridizednanoparticle-bioconjugate probes) are necessary to observe the colorchange. The color change may be quantitated by recording the plate imagewith an optical scanning device such as a flatbed scanner or CCD camera,and analyzing the amount and type of color of each individual spot.Alternatively, a color filter (e.g. red filter) may be used to filterout specific colors so that the signal intensity of each spot may berecorded and analyzed.

[0081] An alternate method for easily visualizing the assay results isto spot a sample of nanoparticle probes hybridized to a target nucleicacid on a glass fiber filter (e.g., Borosilicate Microfiber Filter, 0.7micron pore size, grade FG75, for use with gold nanoparticles 13 nm insize), while drawing the liquid through the filter. Subsequent rinsingwith water washes the excess, non-hybridized probes through the filter,leaving behind an observable spot comprising the aggregates generated byhybridization of the nanoparticle probes with the target nucleic acid(retained because these aggregates are larger than the pores of thefilter). This technique may provide for greater sensitivity, since anexcess of nanoparticle probes can be used.

[0082] Some embodiments of the method of detecting nucleic acid utilizea substrate. By employing a substrate, the detectable change (thesignal) can be amplified and the sensitivity of the assay increased.

[0083] Any substrate can be used which allows observation of thedetectable change. Suitable substrates include transparent solidsurfaces (e.g., glass, quartz, plastics and other polymers), opaquesolid surface (e.g., white solid surfaces, such as TLC silica plates,filter paper, glass fiber filters, cellulose nitrate membranes, nylonmembranes), and conducting solid surfaces (e.g., indium-tin-oxide(ITO)). The substrate can be any shape or thickness, but generally willbe flat and thin. Preferred are transparent substrates such as glass(e.g., glass slides) or plastics (e.g., wells of microtiter plates).

[0084] In one embodiment oligonucleotides are attached to the substrate.The oligonucleotides can be attached to the substrates as described in,e.g., Chrisey et al., Nucleic Acids Res., 24, 3031-3039 (1996); Chriseyet al., Nucleic Acids Res., 24, 3040-3047 (1996); Mucic et al., Chem.Commun., 555 (1996); Zimmermann and Cox, Nucleic Acids Res., 22, 492(1994); Bottomley et al., J. Vac. Sci. Technol. A, 10, 591 (1992); andHegner et al., FEBS Lett., 336, 452 (1993).

[0085] The oligonucleotides attached to the substrate have a sequencecomplementary to a first portion of the sequence of a nucleic acid to bedetected. The nucleic acid is contacted with the substrate underconditions effective to allow hybridization of the oligonucleotides onthe substrate with the nucleic acid. In this manner the nucleic acidbecomes bound to the substrate. Any unbound nucleic acid is preferablywashed from the substrate before adding nanoparticle-bioconjugateprobes.

[0086] Next, the nucleic acid bound to the substrate is contacted with afirst type of nanoparticles having bioconjugates, such asoligonucleotides, attached thereto. The oligonucleotides have a sequencecomplementary to a second portion of the sequence of the nucleic acid,and the contacting takes place under conditions effective to allowhybridization of the oligonucleotides on the nanoparticles with thenucleic acid. In this manner the first type of nanoparticles becomebound to the substrate. After the nanoparticle-oligonucleotideconjugates are bound to the substrate, the substrate is washed to removeany unbound nanoparticle-oligonucleotide conjugates and nucleic acid.

[0087] The oligonucleotides on the first type of nanoparticles may allhave the same sequence or may have different sequences that hybridizewith different portions of the nucleic acid to be detected. Whenoligonucleotides having different sequences are used, each nanoparticlemay have all of the different oligonucleotides attached to it or,preferably, the different oligonucleotides are attached to differentnanoparticles. Alternatively, the oligonucleotides on each of the firsttype of nanoparticles may have a plurality of different sequences, atleast one of which must hybridize with a portion of the nucleic acid tobe detected.

[0088] The first type of nanoparticle-oligonucleotide conjugates boundto the substrate is optionally contacted with a second type ofnanoparticles having oligonucleotides attached thereto. Theseoligonucleotides have a sequence complementary to at least a portion ofthe sequence(s) of the oligonucleotides attached to the first type ofnanoparticles, and the contacting takes place under conditions effectiveto allow hybridization of the oligonucleotides on the first type ofnanoparticles with those on the second type of nanoparticles. After thenanoparticles are bound, the substrate is preferably washed to removeany unbound nanoparticle-oligonucleotide conjugates.

[0089] The combination of hybridizations produces a detectable change.The detectable changes are the same as those described above, exceptthat the when second type of conjugates, multiple hybridizations resultin an amplification of the detectable change. In particular, since eachof the first type of nanoparticles has multiple oligonucleotides (havingthe same or different sequences) attached to it, each of the first typeof nanoparticle-oligonucleotide conjugates can hybridize to a pluralityof the second type of nanoparticle-oligonucleotide conjugates. Also, thefirst type of nanoparticle-oligonucleotide conjugates may be hybridizedto more than one portion of the nucleic acid to be detected. Theamplification provided by the multiple hybridizations may make thechange detectable for the first time or may increase the magnitude ofthe detectable change. This amplification increases the sensitivity ofthe assay, allowing for detection of small amounts of nucleic acid.

[0090] If desired, additional layers of nanoparticles can be built up bysuccessive additions of the first and second types ofnanoparticle-oligonucleotide conjugates. In this way, the number ofnanoparticles immobilized per molecule of target nucleic acid can befurther increased with a corresponding increase in intensity of thesignal.

[0091] In one embodiment for detection of non-nucleic acid analytes (seefor example U.S. patent application Ser. No. 09/820,279, filed Mar. 28,2001, and International application PCT/01/10071, filed Mar. 28, 2001,each of which is incorporated herein by reference) the analyte may bebound directly or indirectly, via covalent or non-covalent interactions,to a substrate. The substrates are rthe same type as described above.For indirect binding, the analyte can be bound to the substrate via alinker, e.g., an oligonucleotide or other spacer molecule.Alternatively, the analyte may be modified by binding it to anoligonucleotide having a sequence that is complementary to at least aportion of the sequence of a capture oligonucleotide bound to asubstrate. The nanoparticle-probe having a recognition group for theanalyte is then contacted with the substrate under conditions effectiveto allow the specific binding of the nanoparticle-probe to the analytebound to the substrate and the presence of the analyte can be visuallydetected either by formation of a spot on the substrate or through theuse of staining material such as silver on gold stain. See FIG. 1 forexamples of this detection method.

[0092] In another method for detecting analytes, the target analyte canbe modified by attaching the analyte to the nanoparticle-probe as therecognition portion of the probe. Thereafter, the modifiednanoparticle-probe is contacted with a substrate having a second memberof the recognition couple bound thereto. The presence of the analyte canbe visually detected either by formation of a spot on the substrate orthrough the use of staining material such as silver on gold stain.

[0093] In yet another method for detecting analytes, the target analyteis modified by binding it to an oligonucleotide having a sequence thatis complementary to at least a portion of a sequence of anoligonucleotide (recognition group) bound to the nanoparticle-probe. Themodified target is then coupled to the nanoparticle-probe by contactingthe modified target and the nanoparticle-probe under conditionseffective for hybridization between the oligonucleotide bound to thetarget and the oligonucleotide bound to the nanoparticle-probe. Thehybridized complex is then contacted with a substrate having arecognition group for the analyte bound thereto. The presence of theanalyte can be visually detected either by formation of a spot on thesubstrate or through the use of staining material such as silver on goldstain. See FIG. 2 for an example of this method.

[0094] When a substrate is employed, a dectectable change can beproduced or enhanced by staining. Staining material, e.g., gold, silver,etc., can be used to produce or enhance a detectable change in any assayperformed on a substrate, including those described above. For instance,silver staining can be employed with any type of nanoparticles thatcatalyze the reduction of silver. Preferred are nanoparticles made ofnoble metals (e.g., gold and silver). See Bassell, et al., J. CellBiol., 126, 863-876 (1994); Braun-Howland et al., Biotechniques, 13,928-931 (1992). If the nanoparticles being employed for the detection ofanalyte do not catalyze the reduction of silver, then silver ions can becomplexed to the target analyte to catalyze the reduction. See Braun etal., Nature, 391, 775 (1998). Also, silver stains are known which canreact with the phosphate groups on nucleic acids.

[0095] An alternate method for utilizing the polythiol nanoparticleprobes is in the application to micro arrays for detecting a variety ofbiomolecules such as nucleic acids, proteins or carbohydrates. Onespecific example is the application of polythiol modifiedoligonucleotide labeled gold nanoparticle probes to the detection ofnucleic acids in a sandwich assay format as described in U.S. Pat. No.6,361,944. In this method, the gold nanoparticle labels are detected viaa silver deposition process. Alternatively, a gold nanoparticledevelopment procedure may be used as described in U.S. Pat. No.6,417,340 and detected optically. It should be noted that the polythiolnanoparticle probes described herein also can be applied as detectionprobes for use as in situ hybridization labels or expanded to otherDNA/RNA detection technologies.

[0096] The invention further provides a kit for performing the assaysfor detecting or quantitating analytes. The kit comprises a containerholding nanoparticle probes having recognition groups attached to them.The kit may also contain other reagents and items useful for performingthe assays. The reagents may include controls, standards, PCR reagents,hybridization reagents, buffers, etc. Other items which be provided aspart of the kit include reaction devices (e.g., test tubes, microtiterplates, solid surfaces (possibly having a capture molecule attachedthereto), syringes, pipettes, cuvettes, containers, etc.

[0097] The following examples are illustrative of the invention but donot serve to limit its scope.

SYNTHESIS EXAMPLES Example 1 Introduction of Amino Groups

[0098] In this example, amino modifier C₆-DT groups are introduced intoan oligonucleotide using an amino modifier phosphoramidite (availablefrom Glen Research, Sterling, Va.) (FIG. 3).

[0099] Protocol. The amino modifier C₆-dT reacts in a manner identicalto normal phosphoramidites, i.e., standard automated oligonucleotidesynthesis. The trifluoroacetyl (TFA) protecting group on the primaryamine is removed during standard ammonium hydroxide deprotection.However, a minor side reaction during ammonia deprotection can lead toirreversibly capping 2-5% of the amine. To prevent this reaction, thesynthesis is carried out using acetyl-protected dC and deprotection iscarried out in 30% ammonia/40% methylamine 1:1 (AMA) at 65° C. for 15minutes.

Example 2 Introduction of Thiol Groups

[0100] In this example, the amine containing oligonucleotide prepared inExample 1 is reacted with 2-iminothiolane.HCI (Traut's Reagent,available from Pierce Chemical Company, Rockford, Ill.) to introducethiol groups (FIG. 4).

[0101] Protocol. The amine containing oligonucleotide prepared inExample 1 is first purified by reverse phase HPLC and is then dissolvedin 50 mM triethanolamine-HCl buffer of pH 8 (or other pH 8 buffer suchas 0.16 M Borate of 10 mM phosphate). A 2-10 fold molar excess of2-iminothiolane-HCl is added. The solution is incubated for 20-60minutes at 0-25° C. The thiolated amine oligonucleotide is thenseparated from the amine oligonucleotide using reverse-phase HPLC (0.03M TEAA buffer (pH 7) with a 1%/min gradient of 95:5 acetonitrile/0.03 MTEAA (pH 7)).

[0102] Alternatively, the following protocol can be used. The aminecontaining oligonucleotide is first purified by reverse-phase HPLC.After purification, the oligonucleotide is re-dissolved in a phosphateor borate buffer (pH 7.2-8.5) containing a 10-100 fold excess of watersoluble carbodiimide (WSC, e.g., ethyl dimethylaminopropyl-carbodiimide)and a 10-100 fold excess of 3-mercaptopropionic acid and allowed tostand for 2-4 hours at room temperature. Next, the oligonucleotide ispurified through a NAP-10 column to remove excess reagents and eluted inwater, followed by reverse-phase HPLC purification.

Example 3 Alternative Protocol for Introduction of Thiol Groups

[0103] This example presents an alternative method for introducing thiolgroups into an oligonucleotide. An amine reactive bifunctionalcrosslinker, e.g., glutaraldehyde, and a heterobifunctional group suchas an alkyl thiol amine are reacted with the amine functionalizedoligonucleotide prepared in Example 2, to create thiol groups (FIG. 5).

[0104] Protocol. The amine containing oligonucleotide is first purifiedbe reverse-phase HPLC. After purification, the oligonucleotide isredissolved in a phosphate or borate buffer (pH 6-9) containing 10%glutaraldehyde and allowed to stand for 1-2 hours. Next, theoligonucleotide is purified through a NAP-10 column to remove excessglutaraldehyde and eluted in pH 6-9 phosphate or borate buffer. A 10-100fold excess of mercaptoethylamine is then reacted with theoligonucleotide for 2-4 hours at room temperature, followed by additionof sodium cyanoborohydride to create a 10% solution for 5 min to reducethe Schiff base. The oligonucleotide is subsequently purified byreverse-phase HPLC.

Example 4 Alternative Protocol for the Introduction of Thiol GroupsUsing a Thiol Functionalized Phosphoramidite

[0105] In this example, a phosphoramidite of formula III (FIG. 6)containing an alkyl thiol or other thiol based functionality issynthesized according to the method of Glick et al., Tetrahedronletters, 1993, 34, 5549-5552, which is incorporated herein in itsentirety. The phosphoramidite is then used to incorporate thiol groupsinto an oligonucleotide using standard phosphramidite methodology.

Example 5 Alternative Protocol for the Introduction of Thiol Groups

[0106] An alternative protocol for the introduction of thiol groups intoan oligonucleotide is as follows. Carboxy-dT (available from GlenResearch, Sterling, Va.) is introduced into an oligonucleotide in ananalogous manner to Example 1. Deprotection is carried out using milddeprotection: 0.4 M methanolic sodium hydroxide (methanol:water 4:1) for17 hours at room temperature. The support is pipetted off and thesolution neutralized with 2 M TEAA. DNA is purified by reverse phaseHPLC. The DNA is resuspended in 100 mM MES buffer (pH 6), water solublecoupling reagents are added (ethyl dimethylaminopropyl-carbodiimide(EDC) and -N-hydroxysulfosuccinimide (sulfo NHS)) at a finalconcentration of 2 mM EDC and 5 mM sulfo-NHS, and the mixture incubatedat room temperature for 15 min. Next, a thiol coupling reagent (e.g.,SH(CH₂)_(x)NH₂) is added at a 10 fold excess and the mixture incubatedat room temperature for 3 hours. The oligonucleotide is purified througha NAP-10 column to remove excess reagents, followed by HPLCpurification.

Example 6 Attachment of the Functionalized Nucleotide to a Nanoparticle

[0107] In this example the thiol functionalized oligonucleotide preparedby any of the methods disclosed herein is attached to a goldnanoparticle through at least two of the thiol groups.

[0108] Protocol. A 4 μM solution of a polythiol modified oligonucleotideis incubated with an approximately 15 nM gold particle dispersion andthen the particles isolated by centrifugation.

Example 7 Synthesis of Epiandrosterone Disulfide Derivative (EPI)Modified Oligonucleotides

[0109] Epiandrosterone can be used as an additional linking element. Itsadvantages include that it is a readily available, easily derivatized toa ketoalcohol and, as a substituent with a large hydrophobic surface,may help screen the approach of water soluble molecules to the goldsurface (Letsinger, et al., J. Am. Chem. Soc. 115, 7535-7536Bioconjugate Chem. 9, 826-830). Incorporation of the epi disulfide intoan oligonucleotide is conducted by phosphoramidite chemistry, asdescribed below.

[0110] The epi disulfide derivative has the structure:

[0111] (a) Synthesis of epi disulfide (FIG. 7). A solution ofepiandrosterone (0.5 g), 1,2-dithiane-4,5-diol (0.28 g), andp-toluenesulfonic acid (15 mg) in toluene (30 mL) was refluxed for 7 hunder conditions for removal of water (Dean Stark apparatus); then thetoluene was removed under reduced pressure and the reside taken up inethyl acetate. This solution was washed with water, dried over sodiumsulfate, and concentrated to a syrupy reside, which on standingovernight in pentane/ether afforded compound epi disulfide 1a as a whitesolid (400 mg); Rf (TLC, silica plate, ether as eluent) 0.5; forcomparison, Rf values for epiandrosterone and 1,2-dithiane-4,5-diolobtained under the same conditions are 0.4, and 0.3, respectively.

[0112] Recrystallization from pentane/ether afforded a white powder, mp110-112° C.; ¹H NMR, δ 3.6 (1H, C³OH), 3.54-3.39 (2H, m 20CH of thedithiane ring), 3.2-3.0 (4H, m 2CH₂S), 2.1-0.7 (29H, m steroid H); massspectrum (ES⁺) calcd for C₂₃H₃₆O₃S₂ (M+H) 425.2179, found 425.2151.Anal. (C₂₃H₃₇0₃S₂)S: calcd, 15.12; found, 15.26.

[0113] (b) Preparation of Steroid-Disulfide Ketal PhosphoramiditeDerivative (FIG. 7)

[0114] Epi disulfide la (100 mg) was dissolved in THF (3 mL) and cooledin a dry ice alcohol bath. N,N-diisopropylethylamine (80 μL) andβ-cyanoethyl chlorodiisopropylphosphoramidite (80 μL) were addedsuccessively; then the mixture was warmed to room temperature, stirredfor 2 h, mixed with ethyl acetate (100 mL), washed with 5% aq. NaHCO₃and with water, dried over sodium sulfate, and concentrated to dryness.The residue was taken up in the minimum amount of dichloromethane,precipitated at −70° C. by addition of hexane, and dried under vacuum;yield 100 mg; ³¹P NMR 146.02.

Example 8 Preparation of 5′-Modified Oligonucleotides (FIG. 8)

[0115] 5′-Modified oligonucleotides are constructed on CPG supportsusing conventional phosphoramidite chemistry, except that compound 1b isemployed in the final phosphitilation step. Products are cleaved fromthe support by treatment with concentrated NH₄OH for 16 h at 55° C. Theoligonucleotides 1c are purified by reversed phase HPLC on a DionexDX500 system equipped with a Hewlett Packard ODS Hypersil column(4.6×200 nm, 5 μm particle size) using TEAA buffer (pH 7.0) and a 1%/mingradient of 95% CH₃CN/5% 0.03 TEAA at a flow rate of 1 mL/min.

Example 9 Further Thiol Functionalization of 1c by Introduction ofFluorinated Nucleotides and Conversion to Thiol Linked Nucleotides

[0116] In this example, fluorine modified Inosine nucleotides (2-F-dI;available from Glen Research, Sterling, Va.) are introduced intooligonucleotide 1c and treated with cystamine after completingsynthesis. See L. V. Nechev, I. Kozekov, C. M. Harris, and T. M. Harris,Chem Res Toxicol, 2001, 14, 1506-1512; A. R. Diaz, R. Eritja, and R. G.Garcia, Nucleos Nucleot, 1997, 16, 2035-2051; D. A. Erlanson, J. N. M.Glover, and G. L. Verdine, J. Amer. Chem. Soc., 1997, 119, 6927-6928.

[0117] Protocol. 1. Incorporate 2-F-dI at the desired position usingstandard synthesis conditions with 2-F-dI-CE phosphoramidite(commercially available form Glen Research). 2. Nucleoside Conversion:At the conclusion of oligonucleotide synthesis, rinse the synthesiscolumn with acetonitrile and roughly dry the support with argon.Dissolve desired primary amine in 1-2 mL DMSO (other organic solvent canbe substituted depending on solubility of amine) at a concentration of0.5 M. Treat the support in the column with the amine solution using two1 mL disposable syringes. Incubate for 18-24 hours at RT to effectconversion. An alternate approach is to transfer the support to aSarstedt tube, add the amine solution and incubate as above. 3.Preliminary O6 Deprotection: Wash the support 2 times with DMSO then 3times with acetonitrile and roughly dry the support with argon. Usingtwo disposable syringes as above, treat the support two times with 1 mleach 1M DBU in acetonitrile one hour each time. Rinse the support with 1ml each of methanol X 2, and acetonitrile X 3. Roughly dry the supportwith argon and deprotect the oligo 1d from the support using ammoniumhydroxide as normal.

STABILITY EXAMPLES

[0118] Stability of the nanoparticle probes of the invention wasevaluated by spotting the subject mixtures on a solid white surface(such as a C-18 silica TLC plate or a reversed phase (RP) HPLC plate)and observing the color of the spot after drying. Red indicates startingnanoparticle with DNA strands on dispersed in solution, and violetindicates particle aggregation due to partial displacement ofoligonucleotides on the gold nanoparticle, and blue indicates evengreater particle aggregation due to more extensive displacement ofoligonucleotides.

Example 10 EPI+2S Probes

[0119] Oligonucleotides having the sequence 5′-Epi-SH-SH-al 8-gcg gaagaa tgt gtc-3′ [SEQ ID NO:1] were prepared as described in Examples 7-9.These oligonucleotides have an analogous structure to oligonucleotides1d in FIG. 7, i.e., they possess an epi disulfide moiety and two furtherthiol groups on the backbone. The oligonucleotides are loaded onto goldnanoparticles as described below. The probes are representative ofprobes of the invention and are denoted “EPI+2S probes.”

[0120] EPI+2S probes are prepared (loaded) in two different saltsolutions; the same oligonucleotide is loaded in either 0.85 M sodiumchloride or in 2.2 M sodium chloride. The EPI-2S probes' length is 35mertotal and contains no fillers.

[0121] Attachment of Oligonucleotides to Gold Nanoparticles

[0122] A colloidal solution of citrate stabilized gold nanoparticles(about 10 nM), prepared by the citrate reduction method (Grabar et. al,Anal. Chem. 1995, 67, 735.), was mixed with sulfur modified-a₂₀-probeoligonucleotide (4 μM), and allowed to stand for 24 hours at roomtemperature in 1 ml Eppendorf capped vials. Then, Step 1: 100 μL of a0.1 M sodium hydrogen phosphate buffer, pH 7.0, and 100 μL of 1.0 M NaClwere premixed and added to the solution and allowed to stand for anadditional 12 hours. Step2: Then the salt concentration was increased to0.3M NaCl and kept further 12 h at room temperature. Step 3: At thispoint the salt concentration was increased to 0.85 and kept another 16 hat room temperature. Total salt aging process took 40 h. In the case ofthe 2.2 M salt concentration, work up at the stage of step 3 salt wasincreased gradually to 2.2 M NaCl and kept at room temperature.

[0123] The solution was next centrifuged at 14,000 rpm in an EppendorfCentrifuge 5414 for about 15 minutes to give a very pale pinksupernatant containing most of the oligonucleotide (as indicated by theabsorbance at 260 nm) along with 7-10% of the colloidal gold (asindicated by the absorbance at 520 nm), and a compact, dark, gelatinousresidue at the bottom of the tube. The supernatant was removed, and theresidue was re suspended in the desired buffer.

Example 11 EPI Probes

[0124] Oligonucleotides having an analogous structure to oligonucleotide1c, i.e., possessing an epi disulfide linkage but no other sulfurgroups, were prepared as described in Examples 7-8. The probe length is18mer+A20 linker and total 38mer. The probe sequence is 5′-Epi-a20-cctcaa aga aaa g-3′ [SEQ ID NO:2] and A20-Epi filler. The probe is loadedin 0.85 M NaCl solution. These probes are representative of prior artprobes in that they do not contain additional thiol functionalization ofthe oligonucleotide backbone. The probes are denoted “EPI probes.”

[0125] Attachment of Oligonucleotides to Gold Nanoparticles:

[0126] A colloidal solution of citrate stabilized gold nanoparticles(about 10 nM), prepared by the citrate reduction method (Grabar et. al,Anal. Chem. 1995, 67, 735.) was mixed with sulfur modified-a₂₀-probeoligonucleotide and corresponding sulfur modified-da₂₀ filleroligonucleotide (each to a concentration of 1.7 μM), prepared asdescribed in part B, and allowed to stand for 24 hours at roomtemperature in 1 ml Eppendorf capped vials. Then, Step 1: 100 μL of a0.1 M sodium hydrogen phosphate buffer, pH 7.0, and 100 μL of 1.0 M NaClwere premixed and added to the solution and allowed to stand for anadditional 12 hours. Step2: Then salt concentration was increased to0.3M NaCl and kept further 12 h at room temperature. Step 3: At thispoint salt concentration was increased to 0.85 and kept another 16 h atroom temperature. Total salt aging process took 40 h.

[0127] The solution was next centrifuged at 14,000 rpm in an EppendorfCentrifuge 5414 for about 15 minutes to give a very pale pinksupernatant containing most of the oligonucleotide (as indicated by theabsorbance at 260 nm) along with 7-10% of the colloidal gold (asindicated by the absorbance at 520 nm), and a compact, dark, gelatinousresidue at the bottom of the tube. The supernatant was removed, and theresidue was resuspended in the desired buffer.

Example 12 Binding of EPI+2S to a Target

[0128] This example verified that EPI+2S probes, like EPI probes, bindto a target.

[0129] To 100 μl of a colloid mixture (50 μl of EPI+2S probes and 50 μlof EPI probes), 1 μl of a 10 μM solution of the target (MTHFR 87 mersynthetic target) was added and the sample frozen at −70° C. for 1minute and then thawed at room temperature. After the sample was broughtto room temperature, 3 μl aliquots were spotted on a RP HPLC plate andsolvents evaporated. Simultaneously, control solution prepared in asimilar manner but without target was spotted on the HPLC plate. Afterevaporation of the solvents, the target containing spot turnedcompletely blue, indicating hybridizing to the target. The control spotturned red, indicating absence of hybridization. See FIG. 10. FIG. 10shows binding of the oligonucleotide to a target. In the figure, Rindicates red, B indicates blue and P indicates purple. The target inthis example has the sequence 87mer target: 5′-ggt gtc tgc ggg agc cgattt cat cat cat cac gca gct ttt ctt tga ggc tga cac att ctt ccg ctt tgtgaa ggc atg cac cga-3′ [SEQ ID NO:3].

Example 13 Stability in the Presence of DTT

[0130] This example shows the increased stability of EPI+2S probes inDTT solution, in comparison to EPI probes. The example also shows thatEPI+2S probes prepared in 2.2 M NaCl solution (“2.2 M EPI+2S probes”)are more stable than EPI+2S probes prepared in 0.85 M NaCl solution(“0.85 M EPI+2S probes”).

[0131] To 50 μl of the probe colloid in 0.1 M NaCl and 10 mM Phosphatebuffer at pH 7, 5 μl of 0.1M DTT solution was added and the mixturespotted on a C-18 RP silica plate. The EPI probe spot turned blue in 44h. The 0.85 M loading EPI+2S probe spot turned blue within 72 h,indicating greater stability than the EPI probes. As expected, the 2.2 Mloading EPI+2S probe spot did not turn blue even after 83 h in 0.1M NaClconditions, indicating even greater stability. See FIGS. 11-13. FIGS.11-13 show displacement of the oligonucleotide from the nanoparticle(indicated by a blue spot).

EXAMPLE 14 Stability in the Presence of DTT at Elevated Temperature

[0132] This example reveals the increased stability of EPI+2S probes inthe presence of DTT at elevated temperature, compared with EPI probes.

[0133] To 50 μl of the colloid, 5 μl of 0.1M DTT solution was added andincubated at 60° C. and spotted on a C-18 RP silica plate. The EPI probeturned blue in 70 min, whereas the 0.85 M EPI-2S probe started turningblue at 120 min. See FIG. 14. FIG. 14 shows displacement of theoligonucleotide from the nanoparticle (indicated by a blue spot).

Example 15 Stability in the Presence of DTT and MgCl₂ at ElevatedTemperature

[0134] This example shows the increased stability of EPI+2S probes inthe presence of DTT and MgCl₂ at elevated temperature, compared with EPIprobes.

[0135] To 50 μl of the colloid, 5 μl of 0.1M DTT solution and 10 mMMgCl₂ [final concentration] were added and the samples incubated at 60°C. The samples were spotted on a C-18 RP silica plate. The EPI probespot turned blue in 25 min while the 0.85 M EPI-2S probe spot startedturning blue at 25 min. The 2.2 M EPI-2S probe spot also started turningblue at 25 min. See FIG. 15. FIG. 15 shows displacement of theoligonucleotide from the nanoparticle (indicated by a blue spot).

1 7 1 34 DNA Artificial sequence synthetic oligonucleotide 1 naaaaaaaaaaaaaaaaaag cggaagaatg tgtc 34 2 34 DNA Artificial sequence syntheticoligonucleotide 2 naaaaaaaaa aaaaaaaaaa acctcaaaga aaag 34 3 87 DNAArtificial sequence synthetic oligonucleotide 3 ggtgtctgcg ggagccgatttcatcatcat cacgcagctt ttctttgagg ctgacacatt 60 cttccgcttt gtgaaggcatgcaccga 87 4 12 DNA Artificial sequence synthetic oligonucleotide 4atcggctaat cg 12 5 17 DNA Artificial sequence synthetic oligonucleotide5 nnnnnatcgg ctaatcg 17 6 17 DNA Artificial sequence syntheticoligonucleotide 6 nnnnnatcgg ctaatcg 17 7 17 DNA Artificial sequencesynthetic oligonucleotide 7 nnnnnatcgg ctaatcg 17

What is claimed is:
 1. A nanoparticle-probe comprising a bioconjugatecoupled to a nanoparticle, wherein the bioconjugate is:

wherein n is 2-100; m is 0-100; X is a nucleotide or modifiednucleotide; Z is a nucleotide, modified oligonucleotide, or polyanion; Qis a recognition group; R is linear or branced C1-C8 alkyl; W is asteroid; and each L is a linker formed by the coupling of two moietiesselected from the group consisting of COOH, NH₂, CHO, F, Cl, Br, I, NCO,NCS, allyl, vinyl, and CH₃CO₂ ⁻, or L is —C(═NH₂Cl)(CH₂)₃ ⁻, wherein thebioconjugate is coupled to the nanoparticle through the sulfur groups.2. The probe of claim 1 wherein Q in the bioconjugate comprises anorganic molecule, an organic or inorganic polymer, a receptor, anucleotide, a nucleoside, a polynucleotide, an oligonucleotide, aprotein, an antibody, a peptide, a carbohydrate, a sugar, a hapten, anucleic acid, an amino acid, a peptide nucleic acid, a linked nucleicacid, a nucleoside triphosphate, a lipid, a lipid bound protein, anaptamer, a virus, a cell fragment, or a whole cell.
 3. The probe ofclaim 1 wherein the recognition group comprises a first and a secondrecognition group, wherein the first recognition group is bound to thesecond recognition group and wherein the second recognition group is amember of a recognition couple that specifically binds to a targetanalyte.
 4. The probe of claim 3 wherein the first and secondrecognition groups comprise a specific binding pair.
 5. The probe ofclaim 4 wherein the binding pair is an antibody-antigen or areceptor-ligand.
 6. The probe of claim 3 wherein the first and secondrecognition groups are oligonucleotides having at least a portion of itssequence complementary to each other.
 7. The probe of claim 1 wherein Xand Z are independently adenine, guanine, cystosine, thymine, uracil, anucleotide derivative, a modified nucleotide, or other molecules thatmay be incorporated into the oligonucleotide synthesis process.
 8. Theprobe of claim 7 wherein X is thymine.
 9. The probe of claim 1 wherein Lis —NH —C(═O)—.
 10. The probe of claim 1 wherein n from bioconjugate Dis
 2. 11. The probe of claim 2 wherein Q is an oligonucleotide.
 12. Theprobe of claim 2 wherein Q is a cDNA
 13. The probe of claim 2 wherein Qis a polynucleotide
 14. The probe of claim 1 wherein the nanoparticle isselected from nanoparticles that have an affinity for thiol groups. 15.The probe of claim 14 wherein the nanoparticle is selected from thegroup consisting of a metal, a semiconductor, a magnetic material,including Au, Ag, Pt, Co, CdSe, CdS, or Si.
 16. The probe of claim 15wherein the nanoparticle is gold.
 17. The probe of claim 16 wherein thenanoparticle has a mean diameter of about 5 nm to about 150 nm.
 18. Amethod for detecting a target analyte comprising: providing ananoparticle probe according to claim 1, wherein the recognition groupon the bioconjugate specifically binds to the target analyte; contactingthe nanoparticle probe and the target analyte under conditions effectiveto specifically bind the target analyte to the recognition group; anddetecting the nanoparticle bound to the target analyte.
 19. The methodof claim 18 wherein the target analyte is a nucleic acid.
 20. The methodof claim 19 wherein binding of the recognition group to the nucleic acidis by hybridization of the recognition group with at least a portion ofthe nucleic acid.
 21. The method of claim 18 wherein the target analyteis an antibody.
 22. The method of claim 20 wherein the contactingconditions include freezing and thawing.
 23. The method of claim 19 or21 wherein the detectable change is a color change observable with thenaked eye.
 24. The method of claim 19 or 21 wherein the nanoparticleprobe is detected with a silver stain.
 25. The method of claim 23wherein the color change is observed on a solid surface.
 26. The methodof claim 18 wherein the nanoparticles are made of gold.
 27. A kitcomprising a container holding the nanoparticle probes of claim 1,wherein the recognition groups on the bioconjugates have specificity orbinding affinity for a portion of a target analyte.
 28. The kit of claim27 wherein Q in the bioconjugate comprises an organic molecule, areceptor, a nucleotide, a nucleoside, a polynucleotide, anoligonucleotide, a protein, an antibody, a peptide, a carbohydrate, asugar, a hapten, a nucleic acid, an amino acid, a peptide nucleic acid,a linked nucleic acid, a nucleoside triphosphate, a lipid, a lipid boundprotein, an aptamer, a virus, a cell fragment, or a whole cell.
 29. Thekit of claim 27 wherein X in the bioconjugate is independently adenine,guanine, cystosine, thymine, uracil, or derivative thereof.
 30. The kitof claim 27 where Z in the bioconjugate is independently adenine,guanine, cystosine, thymine, or uracil, or other molecules that can beincorporated into the oligonucleotide synthesis process.
 31. The kit ofclaim 28 wherein Q is an oligonucleotide.
 32. The kit of claim 28wherein Q is an antibody
 33. The kit of claim 27 wherein thenanoparticle is gold.
 34. The kit of claim 27 wherein the target analyteis a nucleic acid.
 35. A method for preparing the nanoparticle probe ofclaim 1 comprising: (i) preparing a bioconjugate; and (ii) contactingthe bioconjugate with a nanoparticle to form the nanoparticle probe. 36.The method of claim 35 wherein the bioconjugate is bioconjugate (B) step(i) comprises: (a) preparing a complex of formula I, wherein R₂ is COOH,NH₂, CHO, F, Cl, Br, I, NCO, NCS, allyl, or CH₃CO₂ ⁻

(b) reacting complex (I) with a thiolating reagent to provide thebioconjugate of formula (B)


37. The method of claim 35 wherein the bioconjugate is bioconjugate (F)and step (i) comprises: (a) preparing a complex of formula II, whereinR₂ is COOH, NH₂, CHO, F, Cl, Br, I, NCO, NCS, allyl, or CH₃CO₂ ⁻

(b) reacting complex (II) with a thiolating reagent to provide thebioconjugate of formula (F)


38. The method of claims 36 or 37 wherein the thiolating reagent isselected from the group consisting of 2-iminothiolane hydrochloride andSH(CH₂)_(n)Y, wherein n is 1-30 and Y is COOH, NH₂, CHO, F, Cl, Br, I,NCO, NCS, allyl, vinyl or CH₃CO₂ ⁻.
 39. The method of claim 36 whereinR₂ is NH₂ and X is modified with a modifier reagent selected from thegroup consisting of C₆-dT, C₆-dC, and C₂-dT.
 40. The method of claim 38wherein the thiolating reagent is SH(CH₂)_(n)COOH, wherein n is 1-30.41. The method of claim 36 wherein step (b) comprises reacting complex(I) with an amine reactive bifunctional crosslinker and an alkyl or arylthiol amine.
 42. The method of claim 41 wherein the amine reactivebifunctional crosslinker is CHO(CH₂)_(n)CHO wherein n is 1-30, 1,4phenylene diisothiocyanate, 1,6 dihexanoic acid, or 1,6 hexanediisocyanate.
 43. The method of claim 42 wherein n is
 3. 44 The methodof claim 41 wherein the alkyl thiol amine is SH(CH₂)_(n)NH₂.
 45. Themethod of claim 41 wherein the aryl thiol amine is SH(C₆H₄)NH₂.
 46. Themethod of claim 35 wherein step (ii) comprises: contacting thebioconjugate (A) with the nanoparticle in water; adding a salt to thewater to form a salt solution; incubating the bioconjugate andnanoparticle in the salt solution to form a bioconjugate-nanoparticleprobe; and isolating The bioconjugate-nanoparticle probe from the saltsolution.
 47. The method of claim 46 wherein all of the salt is added tothe water in a single addition.
 48. The method of claim 46 wherein thesalt is added gradually over time.
 49. The method of claim 46 whereinthe salt is selected from the group consisting of sodium chloride,magnesium chloride, potassium chloride, ammonium chloride, sodiumacetate, ammonium acetate, a combination of two or more of these salts,one of these salts in a phosphate buffer, and a combination of two ormore these salts in a phosphate buffer.
 50. The method of claim 49wherein the salt is sodium chloride in a phosphate buffer.
 51. Themethod of claim 46 wherein the nanoparticle is selected from the groupconsisting of a metal, a semiconductor, a magnetic material, a colloidalmaterial, ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe, CdTe, In₂S₃,In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, GaAs, GaP, BaTiO₃, Co, Fe₂O₃, core shellparticles and alloyed particles.
 52. The method of claim 51 wherein thenanoparticle is gold.
 53. The method of claim 35 wherein thebioconjugate is bioconjugate (A) and step (i) comprises preparingbioconjugate (A) using a thiol modifier reagent, wherein the thiolmodifier reagent has the structure:

wherein n is 1-10.
 54. The method of claim 53 wherein step (ii)comprises: contacting the bioconjugate (B) with the nanoparticle inwater; adding a salt to the water to form a salt solution; incubatingthe bioconjugate and nanoparticle in the salt solution to form abioconjugate-nanoparticle probe; and isolating thebioconjugate-nanoparticle probe from the salt solution.
 55. The methodof claim 54 wherein all of the salt is added to the water in a singleaddition.
 56. The method of claim 54 wherein the salt is added graduallyover time.
 57. The method of claim 54 wherein the salt is selected fromthe group consisting of sodium chloride, magnesium chloride, potassiumchloride, ammonium chloride, sodium acetate, ammonium acetate, acombination of two or more of these salts, one of these salts in aphosphate buffer, and a combination of two or more these salts in aphosphate buffer.
 58. The method of claim 57 wherein the salt is sodiumchloride in a phosphate buffer.
 59. The method of claim 54 wherein thenanoparticle is selected from the group consisting of a metal, asemiconductor, a magnetic material, a colloidal material, ZnS, ZnO,TiO₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe, CdTe, In₂S₃, In₂Se₃, Cd₃P₂,Cd₃As₂, InAs, GaAs, GaP, BaTiO₃, Co, Fe₂O₃, core shell particles andalloyed particles.
 60. The method of claim 59 wherein the nanoparticleis gold.
 61. The method of claim 20 wherein the contacting conditionsinclude heating.
 62. The method of claim 20 wherein the detectablechange is observed on a solid surface.
 63. A method for detecting anucleic acid having a first and second portion comprising: (a) providinga substrate having attached thereto oligonucleotides complementary tothe first portion of the nucleic acid; (b) providing nanoparticle-probesaccording to claim 1, wherein the nanoparticles have oligonucleotidesbound thereto as recognition group, the oligonucleotides have a sequencethat is complementary to the second portion of the nucleic acid to bedetected; (c) contacting the substrate and nanoparticles with thenucleic acid under conditions effective for hybridization between thenucleic acid, the oligonucleotides bound to the substrate and theoligonucleotides bound to the nanoparticles; and (d) detecting thenanoparticles bound to the nucleic acid bound to the substrate.
 64. Themethod of claim 63 wherein the nucleic acid is contacted with thesubstrate provided in (a) prior to contacting it with the goldnanoparticles provided in (b).
 65. The method of claim 63 wherein thenucleic acid is contacted with the nanoparticles provided in (b) priorto contacting it with the substrate provided in (a).
 66. The method ofclaim 63 wherein the nanoparticle comprises oligonucleotidescomplementary to oligonucleotides on second nanoparticles and form anaggregate with the second gold nanoparticles.
 67. The method of claim 63wherein the nanoparticle is detected with a silver stain.
 68. The methodaccording to claim 63 wherein the nucleic acid is RNA or DNA.
 69. Themethod according to claim 63 wherein the nucleic acid is human,bacterial or viral or fungal origin.
 70. The method according to claim63 wherein the nucleic acid is a gene associated with a disease.
 71. Themethod of claim 63 wherein the nucleic acid is a synthetic DNA, asynthetic RNA, a structurally-modified natural or synthetic RNA, or astructurally-modified natural or synthetic DNA.
 72. The method of claim63 wherein the nucleic acid is a product of a polymerase chain reactionamplification.
 73. The method of claim 63 wherein the substrate has aplurality of oligonucleotides attached to it in an array to allow forthe detection of multiple portions of a single nucleic acid, thedetection of multiple different nucleic acids, or both.
 74. The methodof claim 73, wherein the substrate is a transparent substrate or opaquewhite substrate.
 75. The method of claim 63 wherein the nanopartilcesare made of gold.
 76. The method of claim 63 wherein the substrate iscontacted with staining material to produce the detectable change. 77.The method of claim 76 wherein the staining material is silver stain.78. A method for detecting an analyte in a sample comprising: providinga type of nanoparticle conjugate according to claim 1, the conjugatehaving a recognition group that is a specific binding complement of saidanalyte; contacting the analyte with the nanoparticle conjugate underconditions effective to allow specific binding interactions between theanalyte and specific binding complement bound to the nanoparticleconjugate; and observing a detectable change brought about by thespecific binding interaction of the analyte and the specific bindingcomplement of said analyte.
 79. A method for detecting an analyte in asample comprising: providing a type of nanoparticle conjugate accordingto claim 1, the conjugate having oligonucleotides bound thereto as arecognition group, at least a portion of the oligonucleotides attachedto the nanoparticles are bound, as a result of hybridization, to secondoligonucleotides having bound thereto a specific binding complement ofsaid analyte; contacting the analyte with the nanoparticle conjugateunder conditions effective to allow specific binding interactionsbetween the analyte and specific binding complement bound to thenanoparticle conjugate; and observing a detectable change brought aboutby the specific binding interaction of the analyte and the specificbinding complement of said analyte.
 80. The method according to any oneof claims 78-79, wherein the analyte is polyvalent and binds to two ormore nanoparticle conjugates.
 81. The method according to any one ofclaims 78-79, wherein the analyte is polyvalent and specifically bindsto two or more nanoparticle conjugates.
 82. The method according to anyone of claim 78-79, wherein the contacting conditions include freezingand thawing.
 83. The method according to any one of claims 78-79,wherein the contacting conditions include heating.
 84. The methodaccording to any one of claims 78-79, wherein the detectable change isobserved on a solid surface.
 85. The method according to any one ofclaims 78-79, wherein the detectable change is a color change observablewith the naked eye.
 86. The method according to any one of claims 78-79,wherein the color change is observed on a solid surface.
 87. The methodaccording to any one of claims 78-79, wherein the nanoparticles are madeof gold.
 88. A method for detecting an analyte comprising: providing (i)a support having an analyte bound thereto and (ii) a nanoparticleconjugate according to claim 1, the conjugate having oligonucleotidesbound thereto as a recognition group, a least a portion of theoligonucleotides are bound, as a result of hybridization, to secondoligonucleotides having a specific binding complement of said analyte;contacting the analyte bound to the support to the nanoparticleconjugate under conditions effective to allow specific bindinginteractions between the analyte and specific binding complement boundto the nanoparticle conjugate; and observing a detectable changedependent on the specific binding of the analyte to the specific bindingcomplement of the analyte.
 89. A method for detecting an analytecomprising: providing (i) a support having a oligonucleotides boundthereto, (ii) an analyte having an oligonucleotide bound thereto, theoligonucleotide bound to the analyte has a sequence that iscomplementary to the oligonucleotides bound to the support; and (iii) atype of nanoparticle conjugate according to claim 1, the conjugatehaving oligonucleotides bound thereto as a recognition group, at least aportion of the oligonucleotides bound to the nanoparticle are bound, asa result of hybridization, to second oligonucleotides having boundthereto a specific binding complement of said analyte; contacting theoligonucleotides bound to the support with the olignonucleotide bound tothe analyte under conditions effective to allow hybridization betweenthe oligonucleotides bound to the support and the oligonucleotides boundto the analytes; contacting the analyte bound to the support with thenanoparticle conjugate under conditions effective to allow for specificbinding interactions between the analyte bound to the support and thespecific binding complement bound to the nanoparticle; and observing adetectable change dependent on the specific binding of the analyte tothe specific binding complement of the analyte.
 90. The method of claim89 wherein the substrate is contacted with staining material to producethe detectable change.
 91. The method of claim 90 wherein the stainingmaterial is silver stain.
 92. A method for detecting an analytecomprising: providing (i) a support having an analyte bound thereto and(ii) a nanoparticle conjugate according to claim 1, the conjugateshaving oligonucleotides bound thereto as a recognition group, at leastsome of the oligonucleotides attached to the nanoparticle are bound, asa result of hybridization, to second oligonucleotides having boundthereto a specific binding complement of said analyte; contacting asupport having an analyte bound thereto with the nanoparticle conjugateunder conditions effective to allow specific binding interactionsbetween the analyte bound to the support and specific binding complementbound to the nanoparticle conjugate; contacting the nanoparticleconjugate bound to the support with staining material to produce adetectable change; and observing the detectable change dependent on thespecific binding of the analyte and the specific binding complement ofthe analyte.
 93. The method of claim 63 wherein the substrate has aplurality of oligonucleotides attached to it in an array to allow forthe detection of multiple portions of a single nucleic acid, thedetection of multiple different nucleic acids, or both.
 94. The methodaccording to any one of claims 89 or 92, wherein the detectable changeis observed on a solid surface.
 95. The method according to any one ofclaims 89 or 92, wherein the detectable change is a color changeobservable with the naked eye.
 96. The method according to any one ofclaims 89 or 92, wherein the color change is observed on a solidsurface.
 97. The method according to any one of claims 89 or 92, whereinthe nanoparticles are made of gold.
 98. The method according to any oneof claims 89 or 92, wherein the substrate is a transparent substrate oropaque white substrate.
 99. The method of claim 92 wherein the stainingmaterial is silver stain.